Structural insights into the interaction of human p97 N‐terminal domain and SHP motif in Derlin‐1 rhomboid pseudoprotease

Abstract

The interaction of the rhomboid pseudoprotease Derlin-1 and p97 is crucial for the retrotranslocation of polyubiquitinated substrates in the endoplasmic reticulum-associated degradation pathway. We report a 2.25 Å resolution structure of the p97 N-terminal domain (p97N) in complex with the Derlin-1 SHP motif. Remarkably, the SHP motif adopts a short, antiparallel β-strand that interacts with the β-sheet of p97N-a site distinct from that to which most p97 adaptor proteins bind. Mutational and biochemical analyses contributed to defining the specific interaction, demonstrating the importance of a highly conserved binding pocket on p97N and a signature motif on SHP. Our findings may also provide insights into the interactions between other SHP-containing proteins and p97N.

The AAA+ ATPase p97, a cellular multitool

“…Like other rhomboid proteases, Rhbdl4 is an intramembrane protease, its active site buried in the lipid bilayer where one of its function is the release of growth factors [ 122 ]. The protease has been shown to efficiently form a ternary complex with p97 and K48-linked polyubiquitin [ 123 , 124 ]. A VBM, whose interaction with p97 has been comprehensively characterized biochemically, is located at its C-terminus, next to a ubiquitin-interacting motif [ 123 , 124 ].…”

The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97

“…The initial crystal structure of full-length p97 in complex with the SHP motif of UFD1 revealed that the motif adopts a mostly extended, yet slightly bent conformation, and binds at the periphery of the C-terminal α+β subdomain (Nc, aa 112–186) of the N domain, in direct vicinity of the ND1 linker (Hänzelmann and Schindelin, 2016a). Subsequently, a high-resolution structure of the N domain in complex with the UFD1-SHP using an N domain-SHP fusion protein (Le et al, 2016) (Figures 4B,C) as well as the N domain structure with the SHP of the DERLIN1 (DER1) rhomboid pseudoprotease (Lim et al, 2016b) (Figure 4C) were determined. The SHP motif forms a random coil interrupted by a small two amino acid long β-sheet, which associates with the central four-stranded β-sheet, thereby extending it to a five-stranded antiparallel β-sheet.…”

“…In addition, an adjacent α-helix stabilizes the complex. The motif thus binds in a hydrophobic binding pocket and is stabilized between one of the β-strands and this α-helix, which together are arranged into a β-β-α super-secondary structure, a well-known binding mode mediating protein-protein interactions (Lim et al, 2016b). The two strictly conserved glycine residues (GxG) generate a sharp kink in the middle of the SHP motif, thereby enabling the bending of the motif upon binding to the N domain.…”

“…Bottom, cartoon representation of the overall structure of the UFD1 SHP binding motif (colored in purple) bound to the p97 N domain (β-strands in dark gray and α-helices in light gray) (pdb entry 5B6C; Le et al, 2016). (C) Stick representations of the SHP binding motifs of UFD1 (left, pdb entry 5B6C, colored in purple; Le et al, 2016) and DER1 (right, pdb entry 5GLF, colored in purple; Lim et al, 2016b). The p97 N domain is shown as molecular surface (colored according to hydrophobicity).…”

“…(A) Binding site competition. Superposition of p97 N domain cofactor complexes: SHP of UFD1 (pdb entry 5B6C, colored in purple; Le et al, 2016), VIM of gp78 (pdb entry 3TIW, colored in green; Hänzelmann and Schindelin, 2011), VBM of RHBDL4 (pdb entry 5EPP, colored in light blue; Lim et al, 2016b), FAF1-UBX (pdb entry 3QQ8, colored in gold; Hänzelmann et al, 2011) and NPL4-UBXL (pdb entry 4RV0, colored in olive; Hao et al, 2015). Cofactors are shown in cartoon or stick representation and p97 N as molecular surface (Nn in dark gray, Nc in light gray).…”